Raman imaging of PLGA microsphere degradation inside macrophages

Understanding the degradation behavior of polymeric microspheres is crucial for the successful application of such devices in controlled drug delivery. The degradation mechanism of poly(lactic-co-glycolic acid) (PLGA) microspheres inside phagocytic cells is not known, but different models for degradation in aqueous solution have been proposed. We have used confocal Raman spectroscopy and imaging to study the intracellular degradation of PLGA microspheres inside individual macrophages. Our results show that ingested microspheres degrade in a heterogeneous manner, with a more rapid degradation in the center. Comparison of Raman spectra from degrading beads with those of uningested beads reveals that ester hydrolysis occurs throughout the phagocytosed microspheres, with a selective loss of glycolic acid units. Furthermore, we show that PLGA degradation is a cell-mediated process, possibly caused by the low pH of the phagosome and/or the presence of hydrolytic enzymes. In conclusion, we have demonstrated that the chemical composition of degrading polymers inside cells can be probed by Raman spectral imaging. This technique will expand the capabilities of investigating biomaterial degradation in vivo.     

A review of the bioanalytical methods for the quantitative determination of capecitabine and its metabolites in biological matrices

The bioanalysis of the oral anticancer drug capecitabine and its metabolites has been investigated extensively over the past years. This paper reviews methods for the bioanalysis of capecitabine and its metabolites. The focus of this review will be on sample pre-treatment, chromatography and detection. Furthermore, the choice of standards and analytical problems encountered during analysis of capecitabine and its metabolites in biological matrices will be discussed. The major challenges in the bioanalysis of capecitabine and its metabolites are the simultaneous extraction and analysis due to the differences in polarity of the analytes. Furthermore we evaluate currently described methods for the quantification of capecitabine and its metabolites. Future wishes and perspectives are stated that could serve as an inspiration for further development of assays for the quantification of capecitabine and its metabolites.     

Batch process monitoring using on-line MIR spectroscopy

Many high quality products are produced in a batch wise manner. One of the characteristics of a batch process is the recipe driven nature. By repeating the recipe in an identical manner a desired end-product is obtained. However, in spite of repeating the recipe in an identical manner, process differences occur. These differences can be caused by a change of feed stock supplier or impurities in the process. Because of this, differences might occur in the end-product quality or unsafe process situations arise. Therefore, the need to monitor an industrial batch process exists. An industrial process is usually monitored by process measurements such as pressures and temperatures. Nowadays, due to technical developments, spectroscopy is more and more used for process monitoring. Spectroscopic measurements have the advantage of giving a direct chemical insight in the process. Multivariate statistical process control (MSPC) is a statistical way of monitoring the behaviour of a process. Combining spectroscopic measurements with MSPC will notice process perturbations or process deviations from normal operating conditions in a very simple manner. In the following an application is given of batch process monitoring. It is shown how a calibration model is developed and used with the principles of MSPC. Statistical control charts are developed and used to detect batches with a process upset.     

A Robust Thermal Modulator for Comprehensive Two-Dimensional Gas Chromatography

In comprehensive two-dimensional gas chromatography (GC×GC), two capillary columns are connected in series through an interface known as a “thermal modulator”. This device transforms effluent from the first capillary column into a series of sharp injection-like chemical pulses suitable for high-speed chromatography on the second column. Dramatic increases in the resolving power, sensitivity, and speed of the gas chromatograph result. This paper describes the development of a robust and reliable thermal modulator for GC×GC.     

A high performance liquid chromatographic method for the screening of 17 diuretics in human urine

A simple and adequate HPLC method was developed for screening of human urine for the following 17 diuretic drugs: acetazolamide, bendrofluazide, bumetanide, canrenoic acid, chlorothiazide, chlorthalidone, clopamide, epitizide, etacrynic acid, furosemide, hydrochlorothiazide, indapamide, mefruside, piretanide, spironolactone, torasemide, and triamterene. The assay involves extraction from two 2 mL urine samples with ethyl acetate at pH = 5, washing with a phosphate buffer at pH = 6 and analysis by HPLC using a reversed phase C18 column and ultraviolet detection with a diode array detector for all drugs (except triamterene) using two eluents consisting of water, triethylamine, phosphoric acid and acetonitrile at different ratios and different pH values. Triamterene is determined by direct injection of diluted urine onto the column and is measured by fluorescence detection. The recoveries of the diuretic drugs were determined at two different concentrations and ranged from 43–110% (median: 87%) which is sufficient to detect abuse of these drugs. The repeatability of the assay ranged from 1–12% (median: 5.5%). Received: 13 July 1998 / Revised: 23 October 1998 / Accepted: 29 October 1998     

A high performance liquid chromatographic method for the screening of 17 diuretics in human urine

A simple and adequate HPLC method was developed for screening of human urine for the following 17 diuretic drugs: acetazolamide, bendrofluazide, bumetanide, canrenoic acid, chlorothiazide, chlorthalidone, clopamide, epitizide, etacrynic acid, furosemide, hydrochlorothiazide, indapamide, mefruside, piretanide, spironolactone, torasemide, and triamterene. The assay involves extraction from two 2 mL urine samples with ethyl acetate at pH = 5, washing with a phosphate buffer at pH = 6 and analysis by HPLC using a reversed phase C18 column and ultraviolet detection with a diode array detector for all drugs (except triamterene) using two eluents consisting of water, triethylamine, phosphoric acid and acetonitrile at different ratios and different pH values. Triamterene is determined by direct injection of diluted urine onto the column and is measured by fluorescence detection. The recoveries of the diuretic drugs were determined at two different concentrations and ranged from 43–110% (median: 87%) which is sufficient to detect abuse of these drugs. The repeatability of the assay ranged from 1–12% (median: 5.5%).     

Simultaneous determination of clozapine, norclozapine and clozapine-N-oxide in human plasma by high-performance liquid chromatography with ultraviolet detection.

A reversed-phase high-performance liquid chromatographic (HPLC) method for the simultaneous determination of clozapine and its two major metabolites, norclozapine and clozapine-N-oxide in human plasma has been developed and validated. The isocratic HPLC assay uses a mobile phase consisting of an acetonitril-buffered aqueous solution containing 146 microL of triethylamine and 200 microL of 85% phosphoric acid, adjusted to pH 3.3 with 10% potassiumhydroxide solution (400:600, v/v) at a flow-rate of 0.8 ml/min and a Lichrospher 100 RP-18 reversed-phase column and UV detection at 215 nm. Doxepine was used as the internal standard. Mean recoveries for clozapine, norclozapine, clozapine-N-oxide and doxepine were 95%, 98%, 96% and 94%, respectively, whereas the respective mean repeatability coefficients of variation were 3.4%, 2.7%, 4.3% and 0.9%. Reproducibility coefficients of variation were 1.3%, 1.8%, 3.6% and 0.5%, respectively. The mean correlation coefficient for the linear calibration curve (n = 2) for clozapine and norclozapine at a concentration range of 100-1600 ng/mL was 0.9998 and 0.9997, respectively; for clozapine-N-oxide (20-200 ng/mL) it was found to be 0.9986. The lower limits of quantitation were 12.5 ng/mL, 10 ng/mL and 12.5 ng/mL for clozapine, norclozapine and clozapine-N-oxide, respectively.     

Pore-covered thermoresponsive membranes for repeated on-demand drug release

Reversible on/off-switching of bovine serum albumin (BSA) permeation through a thermoresponsive composite membrane with negligible permeation in the off-state is demonstrated. UV-photografting of poly(N-isopropylacrylamide) onto a poly(ethylene terephthalate) microfiltration membrane results in a hydrogel graft layer on the irradiated side of the membrane only. The amount of hydrogel grafted onto the membrane can be controlled by the amount of crosslinker. Above the lower critical solution temperature (LCST) of the hydrogel (on-state), the shrunken state of the graft layer appears to only partially cover the membrane, allowing BSA permeation through the uncovered pores. Provided the grafting degree is high enough, the swollen hydrogel covers the membrane completely below the LCST (off-state), thus preventing BSA permeation. The on-demand release mechanism proposed here is based on switching the membrane surface coverage rather than previously reported switches based on effective pore size or hydrogel mesh size. The main advantage of our mechanism is that higher fluxes can be achieved in the on-state, since permeation is not limited by pore-narrowing.     

Characterisation of fluorinated copolymers using liquid chromatography coupled on-line to mass spectrometry, with automated data interpretation

A perfluorinated co-polyether was characterised in terms of the number and type of functional end groups present on the molecule. The polymer was separated chromatographically according to the polarity of the polymer end groups and the separation was coupled on-line to an electrospray ionisation time-of-flight mass spectrometer. Negative-mode electrospray ionisation of the relatively non-polar polymer was achieved by post-column addition of a polar constituent to the mobile phase. LC-MS analysis of polydisperse analytes is a highly data intensive technique and manual interpretation of the resulting data can be extremely complicated, especially for the characterisation of copolymers or polymers with end-group distributions. In order to overcome this problem, an automated data-analysis program was developed that allows the user to quickly determine the probability of the presence of a certain molecular compound. The program evaluated data in terms of the possible combinations of monomeric units and end groups that could be combined to make up the mass values present in the mass spectra. Using the program, the polymer can be characterised according to its molar-mass, chemical-composition and functionality-type distributions. A graphical representation of the LC-MS analyses is presented to give a clear overview of the two-dimensional separation. The identification of various end groups on the polymer is also presented graphically, as (a) a histogram (frequency of matches versus time), (b) a two-dimensional plot (masses that match the particular end group combination versus LC retention time) and (c) a plot of average chemical composition versus LC retention time.